1. Field of the Invention
The present invention relates to a transformant for screening of human immunodeficiency virus(“HIV”) inhibitors, more particularly, to a transformant cotransformed with a plasmid expressing HIV nucleocapsid protein and a plasmid containing HIV psi(ψ) nucleotide sequence and β-galactosidase reporter gene, and a method for screening of HIV inhibitors by employing the said transformant.
2. Background of the Invention
HIV, a pathogen causing the aquired immunodeficiency syndrome (“AIDS”), selectively infects crucial immune cells called CD4+ T helper cells and replicates inside the cells. Infection of HIV leads to the lysis of CD4+ T cells resulting from an interaction between viral env glycoprotein and plasma membrane of target cell and a subsequent reproduction of virus particles. Also, the binding of soluble gp120 to CD4+ molecules onto uninfected T cells block interactions of CD4+ T cells with other immune cells. In addition to depleting CD4+ T cells, impaired are function of cytotoxic T cells expressing CD8+, antibody-dependent cytotoxicity, maturation of CD4+ T cells in thymus, interaction between CD4+ cells and class II MHC on antigen presenting cells, and function of macrophages and natural killer cells. Thus, human immune system is gradually deteriorated after HIV infection.
Until now, drugs suppressing HIV replication have been developed, which include reverse transcriptase inhibitors such as AZT(azidothymidine) and ddI(dideoxyinosine), and protease inhibitors. Recently, the researches to develop DNA vaccines employing nucleotide sequence encoding HIV proteins (see: Hinkula J. et al., Vaccine, 15:874–878, 1997; Calarota et al., Lancet, 351:1320–1325, 1998), and live-attenuated HIV vaccines made by deleting the HIV nef gene are being undertaken (see: Kestler and Jeang, Science, 270:1219–1222, 1995; Chakrabarti et al., Proc. Natl. Acad. Sci., USA, 93:9810–9815, 1996).
Since the said drugs are not able to remove the provirus of HIV of which DNA is inserted into the host immune cell chromosome or not able to selectively remove host immune cells containing the provirus, it cannot be excluded that HIV variants arisen by genetic mutation acquire drug resistance or HIV revertants arisen by recombination of attenuated virus vaccine acquire characteristics of pathogenic HIV (see: Berkhout et al., J. Virol., 73:1138–1145, 1999). Furthermore, it has been found that HIV requires not only CD4+ molecule as the receptor on the surface of host cells, but also coreceptors such as ‘T-cell-line-tropic’ CXCR4/fusin coreceptor or ‘macrophage-tropic’ CCR5 coreceptor for its binding and gaining entry of HIV into host cells (see: Feng et al., Science, 272:872–877, 1996). Thus, one approach for drug therapy is to target these coreceptors in an attempt to inhibit binding of virus onto the host cells. Since the normal function of these coreceptors is to bind ‘chemokines (chemotactic cytokines)’ which plays a role in inflammation reaction, serious side effects may be anticipated (see: Murphy, P. M., Ann. Rev. Immunol., 12:593–633, 1994). In view of above situation, there is a need to develop a novel class of HIV inhibitors which do not affect host immunity or physiological activity relating to receptors, and one approach for such drug therapy is to target HIV specific factors required for virus assembly.
When HIV virus particle is assembled, its genomic RNA is selectively packaged into a virion. It is well known that a specific interaction between the region of nucleocapsid (NC) protein and the viral packaging sequence (encapsidation signal), psi(ψ), allows selective packaging of viral genomic RNA. Psi(ψ), located between long terminal repeat(LTR) of 5′-terminal of genomic RNA and gag gene which encodes precursor poly protein (matrix-capsid-nucleocapsid), has 4 stem-loop structures. However, the screening of compounds or compositions which inhibit the specific viral interaction required for packaging is difficult due to the lack of an easy and efficient assay system for such screening.
Under the circumstances, there are strong reasons for exploring and developing a model system which can be used to detect the specific interaction between HIV NC protein and HIV psi(ψ) sequence as in in vivo, for screening of inhibitors against HIV packaging.